PROJECT SUMMARY/ABSTRACT Nephrolithiasis, generally known as kidney stones, is a common disease that affects approximately 30 million Americans. One of the most critical risk factors for kidney stone formation is hypercalciuria, or high levels of urine calcium (Ca2+). The transient receptor potential vanilloid type 5 (TRPV5) channel, which is primarily expressed in the kidney, has been found to be essential for reabsorption of Ca2+ into the blood. Loss or dysfunction of TRPV5 has been shown to severely increase urine Ca2+ levels and the occurrence of kidney stones. TRPV5 is found in the apical membrane of the nephron epithelium and allows Ca2+ reabsorption from the urine along its concentration gradient. In the absence of modulators, TRPV5 has been proposed to be constitutively active. Endogenous modulators such as calmodulin (CaM) and phosphatidylinositol 4,5- bisphosphate (PI(4,5)P2) have been found to stabilize TRPV5 in the closed or open conformation, respectively. Protein kinase C (PKC)-mediated TRPV5 phosphorylation at Ser299 and Ser654 residues has been shown to enhance open probability of the channel. Small molecule antifungals like econazole and miconazole have been demonstrated to inhibit TRPV5. However, molecular details of this channel modulation and gating are poorly understood. Therefore, the goal of this proposal is to utilize cryo-electron microscopy (cryo-EM) in conjunction with biochemical, electrophysiological and computational approaches to uncover the molecular mechanisms of TRPV5 gating. An in-depth investigation of TRPV5 at the atomic level will pave the way for targeted drug discovery for the control and treatment of hypercalciuria and nephrolithiasis.